Cellulose fermentation process

ABSTRACT

In vivo and in vitro cellulose fermentation by cellulose-digesting microorganisms is increased by conducting the fermentation in the presence of a minor amount of a 2-(chloromethyldithio)acetic acid.

BACKGROUND OF THE INVENTION

The effect of chemical additives in microorganism fermentations has beenextensively studied. For example, P. P. Williams et al, App.Microbiology, 11, 517 (1963) describe rumen bacteria and protozoalresponses to insecticide substrate; J. J. O'Connor et al, J. AnimalSci., 33, 662 (1971) describe the in vivo effect of chemical additiveson production of volatile fatty acids by rumen microorganisms; and L. W.Varner et al, J. Animal Sci., 33, 1110 (1971), describe the influence ofammonium salts upon rumen fermentation by steers; and T. W. Dowe et al.,J. Animal Sci., 16, 93 (1957) describe the effect of corn treated withfungicides (N-trichloromethylthio-delta⁴ -tetrahydrophthalimide) on theperformance of fattening steers.

PRIOR ART STATEMENT

U.S. Pat. No. 2,553,778 of Hawley discloses parasiticidalperchloromethylacetic acid esters. U.S. Pat. Nos. 3,442,941, 3,595,915,3,629,313 and 3,718,687 of Emerson et al disclose pesticidalpolyhaloethyl alkanoic acids. Although the compounds of these referencesare structurally related to the compounds of the invention, thecompounds of the references have been found to be ineffective forincreasing the rate of cellulose digestion by microorganisms.

DESCRIPTION OF THE INVENTION

The cellulose-fermentation-accelerating compounds of the invention are2-(chloromethyldithio) acetic acid, 2-(dichloromethyldithio)acetic acidand 2-(trichloromethyldithio)acetic acid. The preferred compound is2-(trichloromethyldithio)acetic acid.

The amount of compound employed in the process of invention depends inpart upon the type of cellulosic material and the particularmicroorganism(s) employed. Generally, weight ratios of compounds tocellulosic matter in the range of about 1:10 to 1:1,000,000 areeffective, although weight ratios in the range of about 1:100 to1:10,000 are preferred.

In in vitro cellulose fermentation processes, the compound is generallyadded directly to the fermentation process. In in vivo cellulosedigestion, the compound may be orally administered to the aminal alongwith the cellulosic feedstuff. Alternatively, the cellulosic feedstuffmay be pretreated with an effective amount of the compound prior tofeeding the animal.

The process of the invention is generally applicable to in vivo or invitro cellulose fermentation by microorganisms. Examples of in vitrocellulose fermentation by microorganisms are the aerobic and/or anerobicdestruction of cellulosic wastes in sewage plants; conversion ofcellulose to sugar by microorganisms such as Trichoderma viride;conversion of cellulose to singlecell proteins by microorganisms such asBacteroidaceae, Cellulomonas and Alcaliginis; and the biodegradation oflignincellulosic plant material. Examples of in vivo fermentation bymicroorganisms are cellulosic digestion by rumen microorganisms ofruminant animals, cecum microorganisms of animal intestines, and othercellulolytic organisms in the alimentary tracts of herbivores.

The process of the invention is suitably employed for all types ofcellulosic material such as paper, municipal waste and plant products,e.g., wood, cotton, straw, bagasse, rice hulls, etc.

EXAMPLES Example 1 -- Cotton digestion by Bacteroides succinogenes

The organism, Bacteroides succinogenes, was obtained from the AmericanType Culture Collection, No. 19169.

    ______________________________________                                                     Bacto-fluid Thioglycollate                                       Nutrient Source:                                                                           (29 g formulation/liter of H.sub.2 O)                            ______________________________________                                                   Bacto-Casitone                                                                              15.0      g                                                     Bacto-Yeast Extract                                                                         5.0       g                                                     Bacto-Dextrose                                                                              5.0       g                                                     NaCl          2.5       g                                                     1-Cystine, Difco                                                                            0.5       g                                                     Thioglycolic Acid                                                                           0.3       ml                                                    Bacto-Agar Resazurin,                                                         Certified     0.001     g                                          ______________________________________                                    

The rate of cotton digestion in the presence of several test compoundsin the above nutrient broth with Bacteroides succinogenes was determinedby the following procedure:

Cotton (100 mg) was placed in screw-cap tubes. To these the testcompound (1 microgram) and the nutrient source (20 ml) were added tocompletely fill the tube.

The tubes were then sterilized, cooled and inoculated with the microbe(1 loop of inoculation needle), their caps tightened, and incubated in awater bath at about 40° C.

The tubes were stirred throughout incubation and the caps loosened every2 hours for the first 18 hours and every 6 hours thereafter to releasegases produced by the fermentation. After 70 hours of incubation, mostof the fermentation processes had subsided, as noted by cessation of gasaccumulation.

After various periods of incubation, the tubes were emptied onpreviously weighed filter paper. The filter paper was washed severaltimes and dried to a constant weight. The weight of the undigestedcotton was determined by difference.

The cellulose digestion results are tabulated in Table I. The resultsare based on the average of 48 runs and standard deviation analysisshowed the results to be significant 1% level.

                  TABLE I                                                         ______________________________________                                                           Cotton                                                     Test Compound      Digestion %                                                                              Acceleration                                    ______________________________________                                        Control            38.6%      --                                              2-(trichloromethyldithio)-                                                                       43.6%      13%                                             acetic acid                                                                   2-(1,1,2,2-tetrachloroethyldithio)-                                                              38.0%      0                                               acetic acid                                                                   ______________________________________                                    

Example 2 -- Cotton digestion by Bacteroides succinogenes

The rate of cotton digestion with Bacteroides succinogenes in thepresence of several test compounds was determined by a procedureidentical to that of Example 1. The test compounds and the results aretabulated in Table II.

                  TABLE II                                                        ______________________________________                                                           Cotton                                                     Test Compound      Digestion %                                                                              Acceleration                                    ______________________________________                                        Control            37.1%      --                                              Methyl 2-(trichloromethyldithio)-                                                                36.2%      0                                               acetate                                                                       Methyl 2-(1,1,2,2-tetrachloroethyl-                                                              36.7%      0                                               dithio)acetate                                                                ______________________________________                                    

Example 3 -- Plant Cellulose Digestion by Bacteroides succinogenes

Lignin-cellulosic matter of herbaceous plant forage was digested byBacteroides succinogenes in a purified medium in the presence of2-(trichloromethyldithio)acetic acid at a concentration of 10micrograms/ml by a procedure identical to that of Example 1. After 70hours incubation, the percent cotton digestion was 54.3%. In anuntreated control run, the percent cotton digestion was 41.1%.

This example exemplifies the in vitro separation of cellulose fromlignin-cellulosic matter by biodegradation of the cellulose.

EXAMPLE 4 -- Cotton Digestion by Ruminococcus albus

Ruminococcus albus was obtained from the America Type CultureCollection. It was cultured on Pseudomonas medium broth which containedthe following (per liter of distilled H₂ O):

    ______________________________________                                        Nitrilotriacetic acid  1.91   g                                               K.sub.2 HPO.sub.4      8.71   g                                               Na.sub.2 SO.sub.4      0.57   g                                               MgSO.sub.4             0.25   g                                               FeSO.sub.4             0.5    mg                                              Ca(NO.sub.3).sub.2     0.5    mg                                              Agar                   1      g                                               ______________________________________                                    

About 20 ml of the medium and 0.1 g cotton were added to each of 48screw-cap tubes and sterilized. The tubes were then inoculated with 1loopful of the microbe. To half of the tubes was added sufficient2-(trichloromethyldithio)acetic acid to give a concentration of 10micrograms per ml. The tubes were then sealed and incubated in a waterbath for 70 hours at 40° C. At the end of the incubation period, theweight of undigested cotton was determined.

The treated tubes (average of 24) gave 29.8% cotton digestion. Thecontrol tubes (average of 24 gave 23.2% control.

Example 5 -- Cotton Digestion by Bacteroides succinogenes in rumen fluid

The rate of cotton digestion in the presence of2-(trichloromethyldithio)acetic acid in sterilized rumen fluid withBacteroides succinogenes was determined by a procedure identical to thatof Example 1. After 70 hours incubation, the percent cotton digestionwas 46.6%. In an untreated control run, the percent cotton digestion was39.8%.

Example 6 -- Solka Floc digestion in rumen fluid

Digestion of Solka Floc was determined using a modification of thetwo-stage digestion procedure (Tilley and Terry, J. Brit. Glassl. Sc.18:104-111, 1963). Substrate (in triplicate) was treated with either 0,40, 60, 80, 100 or 150 ppm of the test compound and incubated withbuffered rumen fluid for 24 hours followed by 24 hours pepsin digestion.In vitro digestibility was measured at end of incubation with bufferedrumen fluid (ruminal digestion) and at end of pepsin digestion (totaldigestion).

Rumen fluid was obtained from donor animal maintained on alfalfa hay -corn grain mineral supplement ration at maintenance plus level ofintake. The test compound was added directly to Solka Floc on dry matterbasis.

                  TABLE III                                                       ______________________________________                                        Level of     Percent Ruminal                                                                              Percent Total                                     Test Compound.sup.1                                                                        Digestibility.sup.2                                                                          Digestibility.sup.2                               ______________________________________                                         0 ppm       10.34          26.50                                             40 ppm       13.96 (35.01)  29.53 (11.43)                                     60 ppm       12.73 (23.11)  30.00 (13.21)                                     80 ppm       13.18 (27.47)  29.93 (12.94)                                     100 ppm      12.91 (24.85)  29.16 (10.04)                                     150 ppm      11.96 (15.67)  28.26  (6.64)                                     ______________________________________                                         .sup.1 Values in parentheses represent percent increase.                      .sup.2 Test Compound added on substrate dry matter basis.                

What is claimed is:
 1. A method for accelerating the rate of cellulosefermentation by cellulose-digesting rumen microorganisms which comprisesconducting said fermentation in the presence of a rate-acceleratingamount of a compound selected from 2-(chloromethyldithio)acetic acid,2-(dichloromethyldithio)acetic acid or 2-(trichloromethyldithio)aceticacid.
 2. The method of claim 1 wherein the compound is2-(trichloromethyldithio)acetic acid.
 3. The method of claim 2 whereinthe fermentation is conducted in vitro.
 4. The method of claim 2 whereinthe cellulose is cellulosic waste products.
 5. The method of claim 2wherein the cellulose is cellulosic animal feed.
 6. The method of claim2 wherein the microorganism is Bacteroides succinogenes or Ruminococcusalbus.
 7. The method of claim 2 which comprises orally administeringsaid compound to ruminant animals for cellulose digestion in the rumenof said animals.
 8. The method of claim 2 wherein the fermentation is invivo.